During the first 30 years of this grant, using X-ray crystallography, we have been able to describe the detailed molecular architecture of HIV-1 reverse transcriptase (RT) in complex with dsDNA, RNA/DNA, and dsRNA. These include structures poised for nucleotide incorporation, catalytic complexes, and post-incorporation structures prior to translocation. All of them represent key states for understanding the comprehensive nature of reverse transcription during HIV infection, and the mechanisms of action of and resistance to nucleoside RT inhibitor (NRTI) drugs. In a collaborative effort, our crystallographic structures enabled the discovery of two non-nucleoside RT (NNRTI) drugs (rilpivirine and etravirine), leading to five licensed anti-AIDS medications. During the previous grant period, major highlights included solving RT structures in complex with a dsRNA initiation complex prior to nucleotide incorporation, RT/DNA with an NNRTI, RT with NRTI drugs, and RT with nucleotide-competing RT inhibitors (NcRTIs) including INDOPY-1. We propose to follow up by determining a series of RT/DNA-RNA/RNA structures to map molecular details of how first-strand DNA synthesis is initiated, with a focus on conformational changes in RT and nucleic acid that accompany the transition from initiation (slow) to processive (fast) DNA synthesis. Similarly, details of second-strand initiation will be probed beginning with a structure of RT with the polypurine-tract RNA primer across from a DNA template. Additional HIV-1 RT structures with drugs and investigational inhibitors will be determined to further understand mechanisms of inhibition and resistance, and the potential for additional sites not currently targeted by anti-AIDS drugs. Although the HIV proteins and enzymes are produced initially as part of Gag-Pol precursor polyproteins, relatively little is known about the detailed structure and function of the immature forms prior to maturation. Recently, we have produced multiple-milligram amounts of the HIV-1 Gag-Pol and Pol precursor polyproteins for the first time. Cryo-EM determination of the HIV-1 Pol polyprotein shows a dimeric structure with RT in a p66/p51 heterodimer-like configuration at its core, suggesting that formation of the mature RT structure is guided from early stages of its morphogenesis. Formation of the RT dimeric core also brings the protease monomers into a dimeric arrangement, thus providing structural insight into the potential role of RT in protease activation during virion maturation. We will pursue further cryo-EM, crystallographic, and biophysical studies of the HIV-1 Pol polyprotein and its complexes with relevant binding partners to provide further structural and biophysical insights that shed light on various aspects of virus assembly and maturation. The proposed work relies on collaborations with Jeffrey DeStefano (Maryland), and Stephen Hughes (NCI- Frederick), Mamuka Kvaratskhelia (Colorado-Denver), Ronald Levy (Temple), Dmitry Lyumkis (Salk), Stefan Sarafianos (Emory), and Gilda Tachedjian (Monash).